Topical composition containing amino acid in combination with either interferon of thymidine derivatives for treating viral or inflammation diseases

ABSTRACT

A medically beneficial preparation is provided which contains amino acids and is applicable for augmenting the anti-viral and anti-inflammatory effects of interferon and thimidine-analogous antiherpetic drugs, for amelioration of psoriatic symptoms, and for treating Herpes virus infections.

This application is the national phase under 35 U.S.C. §371 of prior PCT International Application No. PCT/HU97/00035 which has an International filing date of Jul. 3, 1997 which designated the United States of America, the entire contents of which are hereby incorporated by reference.

Subject of the patent a medically beneficial preparation for external use containing amino acids.

The preparation according to the patent description is advantageously applicable for augmentation of the antiviral and anti-inflammatory effects of interferons and thimidine-analogous antiherpetic drugs, for amelioration of psoriatic symptoms and further, it is an effective drug against Herpes virus infections.

BACKGROUND OF THE INVENTION

Interferons are natural proteins with complex biological activity. Most important of their effects are antiviral, cell proliferation inhibitory and immune response enhancing properties.

These effects are utilised in the human therapy. Interferons have therapeutic use in tumour bearing patients.

Such applications are described in the following publications: J. biol. Regul. Homeostatic Agents, 1 pp 93-99 and 177-182, 1987; Intern. J. Cancer, 1987(Suppl.1.), pp 9-13, 1987; J. Interferon Res., Spec. Issue, 1992 April, pp 109-118.

Interferons are also effective in viral infections as it can be seen in the following publications: Lancet, i, p. 128, 1976; Transplantation Proc., 21, pp 2429-2430, 1989; Interferons in the Treatment of Chronic Virus Infections of the Liver, Pennine Press, Macclesfield, 1990.

They have also been proved beneficial in certrain inflammatory diseases: Neurology, 43, pp 655-661, 1993; J. Interferon and Cytokine Res., 15, pp 39-45, 1995.

The high doses applied for reaching a single therapeutic goal, however, may often provoke numerous unnecessary side effects due to the complex actions of these proteins (J. Rheumatol., 20, pp 83-85, 1992; J. Pediatr., 120(3), pp 429-431, 1992; Clin. Exp. Immunol., 90(3), pp 363-367, 1992).

These side effects are quite often dose-limiting factors in the therapeutic use of interferons.

Typically, interferons are applied in combination for therapeutic purposes in order to decrease the severity of side effects. Several different approximations are applicable for combination therapies such as: decrease in the necessary (loses by complementation with drugs of similar mechanism of action (J. Natl. Cancer Inst., 83 pp 1408-1410, 1991); combination with drugs of antagonistic mechanism of action in order to selectively reduce harmful side effects (J. Biol. Resp. Modifiers, 5, pp 447-480, 1986); selective augmentation of the required therapeutic effect by addition of potentiating components or by application of appropriate physical conditions, e.g. hyperthermia (Proc. Soc. Exp. Biol. Med., 169, pp 413, 1982).

It is known from the publications that effective therapeutic application of thimidine-analogous drugs (deoxyuridine derivatives substituted at the 5 position) with antiherpetic action is seriously limited by that fact that a fast viral resistance develops in response to therapeutic concentrations of these drugs. Viral strains resistant to one given drug show crossresistance to other ones with similar chemical structure. Dose reduction of these drugs--if it could be achieved--would reduce the selection pressure on the viruses, thereby reducing, the frequency of the development of resistant mutants and consequently enhancing the therapeutic value of the known antiherpetic agents.

SUMMARY OF THE INVENTION

The purpose of this invention is to enhance selectively the antiviral effects of interferons and antiherpetic thimidine-analogues in order to be able to decrease the effective therapeutic doses.

It was also intended to develop a drug combination beneficial in herpetic infections and effective in reducing or eliminating skin symptoms of psoriasis.

One aim of the inventions is to produce an ointment and liquid for external use which contains low dose (and, thus, free of side effects) antiviral drugs (interfernon, 5-ethyl-2'-deoxyuridine=EDU, 5-iodo-2'-deoxyuridine=IDU,) combined with components (amino acids) selectively potentiating the antiviral activity and the interferon-mediated inhibition of inflammation.

Our invention is based on the recognition that some amino acids are able to potentiate the antiherpetic effect of the thimidine analogue drugs by a factor of several grades (10² -10⁴ times) and likewise affect the antiviral and anti-inflammatory action of interferons without influencing other biological activities of interferons. Furthermore, the preparations are effective against Herpes viruses and alleviate or eliminate psoriatic symptoms of the skin.

Therefore, the core of the invention is a preparation for external use, containing amino acids, advantageously augmenting antiviral and anti-inflammatory drug actions and being benefical in psoriasis.

The preparation is characterised by its composition, containing one or more of the amino acids listed below -D- or L-aspartic acid (Asp), cysteine (Cys), cystine (cys), glycine (Gly), oxyproline (Opr), serine (Ser), tyrosine (Tyr)- and interferon or thimidine-analogous antiherpetic drugs--preferably uridine-derivaties--as required, furthermore, known pharmaceutical vehicles, preferably solvents, preservatives or known ointment bases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The relationship between titre of the percentage of the untreated control and the amino acid concentration.

FIG. 2: The relationship between titre of the percentage of the untreated control and the amino acid concentration in phases I, II, or III.

FIG. 3: The relationship between virus production and Asp in 5 mg/ml concentration.

FIG. 4: The relationship between virus production and Ser in 5 mg/ml concentration.

FIG. 5: The antiviral activity of different concentrations of EDU in the presence of 5 mg/ml Ser and 250 IU/ml of HuIFN-γ.

The invention is further described by the following examples.

EXAMPLE 1

A sterile solution of native human interferon alpha (HuIFN-α)--preferably from the preparation under trade name EGIFERON--was made in water at a concentration of 50000 international units/ml (IU/ml) under aseptic conditions. The solution also contained an amino acid mixture of D-Asp and L-Ser at a concentration of 5 mg/ml for each. 20 w/v % sucrose was added as a conserving agent.

EXAMPLE 2

A sterile solution of native or recombinant human interferon gamma (HuIFN-γ) was made in water at a concentration of 2500 IU/ml and 15 mg/ml of D-Asp and L-Opr. 20 w/v % sucrose was added as a conserving agent.

EXAMPLE 3

A sterile solution of IDU was made in water at a concentration of 25 μg/ml. 25 mg/ml of D-Asp was dissolved in the above solution. 20 w/v % sucrose was added as a conserving agent.

EXAMPLE 4

A sterile solution of EDU was made in water at a concentration of 25 μg/ml. 1 mg/ml of L-Ser and 500 IU/ml of native or recombinant HuIFN-γ was added to the above solution. 20 w/v % sucrose was used as a conserving agent.

EXAMPLE 5

Doses of HuIFN-α according to example 1. (at an IU/g ratio) were mixed with types and doses of amino acids described in the example 1. into a pharmaceutical ointment base (e.g. unguentum hydrophylicum) under aseptic conditions.

EXAMPLE 6

The active ingredients described in the example 2. were mixed into a vehicle according to example 5. at a ratio shown in the exapmle 2. suitably substituting "g" for "ml".

EXAMPLE 7

Antiviral activity of a HuIFN-α (in a prearranged concentration of 500 IU/ml) was measured on WISH (human amnion epithelia) cells against Vesicular stomatitis virus (VSV) in the presence of different amino acids under different experimental conditions. The antiviral test consists of 3 phases. In the I. phase WISH cells are incubated in 96-well flat-bottom microplates until reaching monolayer stage. 100 μl aliquots of twofold seral dilutions of the HuIFN-α samples are then added to the test cells and are incubated for 20-24 hours at 37° C. in 5% CO₂ atmosphere (phase II.). Finally the IFN-treated cells are infected with a predetermined dose of VSV which can kill 100% of the unprotected WISH cells in 24 hours (phase III.). The reference point of a measurement (the titre of HuIFN-α) is that dilution of the IFN sample which provides protection for 50% of the infected cells at the end of the 24 hours infection period. The effects of different amino acids and different conditions were compared by determining the virtual titres of HuIFN-α samples having identical nominal titres. The measurements were done:

a) in the presence of different amino acids at different concentrations in phase II. (FIG. 1.)

b) in the presence of different amino acids at a concentration of 10 mg/ml in phase I., II. or III. (FIG. 2.)

c) in the presence of differenct amino acid pairs in phase II. at individual concentrations of 5--5 mg/ml (Table 1.)

                  TABLE 1                                                          ______________________________________                                         Antiviral titres of the IFN-α samples treated with amino acid            pairs                                                                          in phase II. Results are given in % of the untreated control. The              synergistic co-operation of Asp-Ser and Ser-Tyr pairs should be noted.         1.sup.st amino                                                                         2.sup.nd amino acid                                                    acid    None    Asp    Cys   Gly  Opr   Ser  Tyr                               ______________________________________                                         None    100                                                                    Asp         152      171                                                       Cys          95      109                                                                                  82                                                  Gly         191      174                                                                                  109                                                                                203                                             Opr         107      167                                                                                  102                                                                                223                                                                                  132                                       Ser         201      468                                                                                  197                                                                                209                                                                                  145                                                                                  222                                 Tyr         113      104                                                                                  104                                                                                166                                                                                  125                                                                                  584                                                                                151                             ______________________________________                                    

d) in the presence of different amino acid pairs in phase III. at individual concentrations of 5--5 mg/ml (Table II.)

                  TABLE II                                                         ______________________________________                                         Antiviral titres of the IFN-α samples treated with amino acid            pairs                                                                          in phase III. Results are given in % of the untreated control. Most            important pairings are: Asp-Ser, Asp-Cys, and the 10 mg/ml doses               of Asp, Cys, Tyr and Opr (Asp-Asp, Cys-Cys, Tyr-Tyr and Opr-Opr                pairs respectively). It also should be noted that phase III. is the most       similar in condition to the natural course of infections: the thera-           peutic drug is present simultaneously with the virus, not preceding it.        1.sup.st amino                                                                         2.sup.nd amino acid                                                    acid    None    Asp    Cys   Gly  Opr   Ser  Tyr                               ______________________________________                                         None    100                                                                    Asp           153                                                                                1722                                                         Cys           193                                                                                542    527                                                   Gly           134                                                                                161    192   143                                             Opr           241                                                                                395    249   157                                                                                 3424                                       Ser           115                                                                                664    322   209                                                                                 383  221                                   Tyr           166                                                                                124    197   114                                                                                 296   99   1568                            ______________________________________                                    

EXAMPLE 8

The changes in the antiviral titres of HuIFN-γ samples upon amino acid (10 mg/ml) applications in phase II. or III. were examined in the system described in the example 7. (Table III.)

                  TABLE III                                                        ______________________________________                                         The effects of different amino acids applied in phase II. or                   III. on the antiviral titre of HuIFN-γ. High potention in                phase III. by Asp, Cys and Tyr, and further, the extremely                     high (180-fold) augmentation by Opr can be seen.                               Amino acids                                                                               Effects in phase II.                                                                        Effects in phase III.                                  ______________________________________                                         None       100          100                                                    Asp                                        2263                                Cys                     53                                                                                                1382                                Gly                     99                                                                                                 184                                Opr               no antiviral activity                                                                        18101                                          Ser                                         598                                Tyr                                        1695                                ______________________________________                                    

EXAMPLE 9

Antiviral activities of different concentrations of IDU were measured on a permanent human tumour cell line (Hep2) against human Herpes simplex type 1. Cells were incubated in Petri dishes until monolayer stage, then were infected with a predetermined dose of the challenge virus which can kill 100% of the unprotected cells in 72 hours. The virus was allowed for 1 hour to be adsorbed on the surface of the cells. Next, a fresh nutritive medium was given to the cells containing the drug in a concentration to be tested and the experimental system was incubated for 72 hours at 37° C. in 5% CO₂ atmosphere. The amount of virus produced in the test system was determined as follows: infected cells were disrupted, centrifuged and the supernatants were collected. Serial 10-fold dilutions were made from the supernatants and 100 μl aliquots were measured on Hep2 monolayers in 96-well flat-bottom microplates and incubated for 72 hours, TCID₅₀ values (dilutions which kill 50% of the test cells at the end of the incubation period) were determined for drug treated samples as well as for untreated controls. The antiviral effects of the drugs were calculated from the differences in virus production. Changes in the antiviral activity were measured in the presence of:

a) Asp in 5 mg/ml concentration (FIG. 3.)

b) Ser in 5 mg/ml concentrtion (FIG. 4.).

EXAMPLE 10

Antiviral activities of different concentrations of EDU were determined in the simultaneous presence of 5 mg/ml Ser and of 250 IU/ml HuIFN-γ (FIG. 5.) as described in the example 9.

EXAMPLE 11

Herpes virus infections (HSV 1, Herpes zoster) and inflammatory skin rushes of different aetiology were treated with an ointment described in the example 5. Patients were chosen on voluntary basis and uncontrolled treatments were carried out. The results obtained are summarised in Table IV.

                                      TABLE IV                                     __________________________________________________________________________                   Treatment                                                                Number of                                                                            (daily applications/                                             Disease cases number of days)                                                                         Results   Notice                                        __________________________________________________________________________     labial herpes                                                                          56 cases/27                                                                          2-3/1-3  57/57 healings in 3                                                                      At 1 person vesicles                          (HSV 1)              rsons                                                                                     days                                                                                            extended to the neck                                           and                                                                                                 breast. Healing in                                        3 days.                                                                                             Ointmemt applied at                                       the                                                                                                 onset prevent the                                                              appearance of                                             symptoms.                                     genital herpes                                                                                        2/7                                                                                   Healing in a week                                                                   Widespread ulcerous                         (HSV 2)                                               infection on the                                          leg.                                          zoster                 2/5                                                                                   5/5 healings in a                                                                    Pain quits in 24 hrs.                                                      week                                                                                            Crusting of vesicles                                           starts                                                                                              in 72 hrs.               postzoster                                                                                         3/2-3                                                                                  6/6 healings in 2-3                                                                  No known recurrences                         neuralgia                                                                                                      days                                                                                            since 2 years.                decubitus                                                                                             3/2                                                                                   2/2 healings in 2                                                                    Livid skin, nonulcerous                                                    days                                                                                               state. No symptoms                                                          developed again at the                                                              treated regions                                           during                                                                                              further exposition                                        (bed-                                                                                               bound state).            acne          2-3/2-57 cases/11                                                                        15/17 healings                                                                                Recurrence frequency                                                    ns                                                                                                  decreased at                                               treated                                                                                             patients.                exanthema                                                                                         3/7s/3                                                                                    4/4 healings in 1                                migrans              rsons                                                                                     week                                           traumatic                                                                                             3/2                                                                                   6/6 healings in 24                                                                  Reddening and                               haematoma                                                                                                      days                                                                                            disappearance instead                                          of                                                                                                  the usual                                                 coloration.                                   pruritus                                                                                              2/2                                                                                   Healing in 2 days                                inflammation                                                                                          3/1                                                                                   2/2 healings in 24                                                                  Diaper dermatitis due to                    due to irritation                                                                                              hrs.                                                                                            adult incontinence.           inflammation of                                                                              1                                                                                       3/2                                                                                   Healing in 2 days                                                                    Sterile inflammation around                surgical wounds                                                                                                                     the sutures.              purulent skin                                                                                         2/1                                                                                   2/2 healings in 1                                                                    Probably some anti-                        inflammation                                                                                                   day                                                                                              bacterial effects are                                         also                                                                                                involved. In 1 case                                       our                                                                                                 treatment followed                                        after 4                                                                                             days ineffective                                          tetracycline                                                                                         treatment.              inflammation of                                                                              1                                                                                       3/1                                                                                   healing in 1 day                                                                        Origin of inflammation                  the outer ear                                                                                                                         unknown. No                                              visible signs                                 cavity                                                 of infection.                                            Treatment by                                                                                         earplugs.               "cold allergy"                                                                          3             3/1                                                                                   3/3 healings in 1 day                            skin rushes due                                                                              4                                                                                    2-3/3                                                                                  4/4 healings in 3                                                                        Allergens were bijoux                    to contact                                                                                                     days                                                                                              necklaces, armbands                                          or                                            allergy                            chromium watchbands.                        psoriasis                                                                                      2-3/2-10                                                                                 517 healings                                                                                    Not all types of disease are                                                               responsive.             __________________________________________________________________________ 

What is claimed is:
 1. A medically beneficial preparation for external use comprising a thymidine-analogous antiherpetic drug; one or more amino acids selected from group consisting of D-aspartic acid, L-aspartic acid, cysteine, cystine, glycine, oxyproline, serine and tyrosine in ar amount sufficient to potentiate the antiviral activity of said thymidine-analogous antiherpetic drug; and optionally pharmaceutical additives for external use.
 2. The preparation of claim 1, wherein the antiherpetic thymidine-analogous drugs are uridine derivatives.
 3. The preparation according to claim 1, wherein the thymidine-analogous antiherpetic drug is 5-ethyl-2'-deoxyuridine or 5-iodo-2'-deoxyuridine.
 4. A medically beneficial preparation for external use comprising a thymidine-analogous antiherpetic drug; one or more amino acids selected from group consisting of D-aspartic acid, L-aspartic acid, cysteine, cystine, glycine, oxyproline, serine and tyrosine in an amount sufficient to potentiate the antiviral activity of said thymidine-analogous antiherpetic drug; and optionally pharmaceutical additives for external use, wherein the preparation is a solution.
 5. A medically beneficial preparation for external use comprising a thymidine-analogous antiherpetic drug; one or more amino acids selected from group consisting of D-aspartic acid, L-aspartic acid, cysteine, cystine, glycine, oxyproline, serine and tyrosine in an amount sufficient to potentiate the antiviral activity of said thymidine-analogous antiherpetic drug; and optionally pharmaceutical additives for external use, wherein the preparation is an ointment.
 6. A medically beneficial preparation for external use comprising interferon; one or more amino acids selected from the group consisting of D-aspartic acid, L-aspartic acid, cysteine, cystine, glycine, oxyproline, serine and tyrosine in an amount sufficient to inhibit the inflammatory effects of interferon; and optionally pharmaceutical additives for external use, wherein the preparation is a solution.
 7. A medically beneficial preparation for external use comprising interferon; one or more amino acids selected from the group consisting of D-aspartic acid, L-aspartic acid, cysteine, cystine, glycine, oxyproline, serine and tyrosine in an amount sufficient to inhibit the inflammatory effects of interferon; and optionally pharmaceutical additives for external use, wherein the preparation is an ointment.
 8. A method of treating psoriasis, which comprises externally applying the preparation of claim 1 to a patient suffering from psoriasis.
 9. A method of treating acne, which comprises externally applying an effective amount of the preparation of claim 1 to a patient suffering from acne.
 10. A method of treating skin inflammation due to irritation, which comprises externally applying an effective amount of the preparation of claim 1 to a patient suffering from skin inflammation due to irritation.
 11. A method of treating nonulcerous decubitus, which comprises externally applying an effective amount of the preparation of claim 1 to a patient suffering from nonulcerous decubitus.
 12. A method of treating skin inflammation due to allergic conditions, which comprises externally applying an effective amount of the preparation of claim 1 to a patient suffering from skin inflammation due to allergic conditions.
 13. A method of treating sterile wound inflammation, which comprises externally applying an effective amount of the preparation of claim 1 to a patient suffering from sterile wound inflammation.
 14. A method of treating herpes viruses, which comprises externally applying an effective amount of the preparation of claim 1 to a patient suffering from herpes virus. 